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Plasmid extraction sans commercial kits


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3 replies to this topic

#1 Julio-Claudian

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Posted 03 March 2009 - 07:14 PM

Dear all,

Can anyone link me to sites with protocols for plasmid extraction without the use of kits?

Commercially, there are kits for the extraction of the genomic DNA and also the plasmid but what separates these two kits? i.e. how is it that one gives the genomic DNA and the other, plasmids?

Cell lysis > Protein precipitation > Transfer into isopropanol > Ethanol > Reconstitute

Thanks a lot!

#2 phage434

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Posted 03 March 2009 - 07:39 PM

Plasmid DNA is separated by physical rather than chemical techniques. The chromosomal DNA consists of long strands that form sticky masses when cells are lysed. A standard prep uses SDS + NaOH for the lysis, followed by neutralization with KAc. The potassium dodecyl sulfate (KDS) which forms is insoluble, and precipitates out as a white solid, taking the stringy chromosomal DNA with it. The short plasmid DNA remains in solution and is separated for further purification. This is not a perfect process, and relies on users not shearing or vortexing the lysed cells. Even so, chromosomal DNA contamination is inevitable.

#3 mikew

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Posted 04 March 2009 - 11:55 AM

Hi,

Your lab must have the "Molecular Cloning" books.
Every lab has them. Or maybe "Current Protocols".
If you do not have them, get them and read them.
EVERY lab in the world has them.
They have great protocols for plasmid purification.
I don't recommend Cesium Chloride because it is time consuming.
I do recommend the Polyetylene Glycol method. Not as pure in general but much
easier.

Here's a link for these books at Amazon. It is a 3 volume set. Get them.
http://www.amazon.co...a...6482&sr=8-1

#4 Julio-Claudian

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Posted 04 March 2009 - 03:29 PM

Thanks phage434. Will do, mikew. Thank you! :o




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